Functionalized rgd peptidomimetics and their manufacture, and implant having a coating containing such functionalized rgd peptidomimetics

ABSTRACT

At least some embodiments of the invention relates to an implant having a coating that contains or is composed of a functionalized RGD peptidomimetic RGD-P1 having the formula (1) and/or a functionalized RGD peptidomimetic RGD-P2 having the formula (2), and an associated manufacturing method.

CROSS REFERENCE

The present application claims priority on U.S. Provisional ApplicationNo. 61/412,804 filed on Nov. 12, 2010, which application is incorporatedby reference herein.

TECHNICAL FIELD

The invention relates to functionalized RGD peptidomimetics and anassociated manufacturing method, and to an implant having a coatingcontaining such RGD peptidomimetics.

BACKGROUND

Implants are utilized in modern medical technology in a variety ofembodiments, including for example implants that perform a supportfunction, such as stents, implants that perform a control function e.g.electrodes and implants that perform a measurement or monitoringfunction (e.g. sensors). Implants can be used for example to supportvessels, hollow organs, and ductal systems (endovascular implants, e.g.stents), to fasten and temporarily fix tissue implants and tissuetransplants in position, as well as for orthopedic purposes such as pin,plate, or screw and others. The stent is a form of an implant that isused particularly frequently.

Stent implantation has become established as one of the most effectivetherapeutic measures for treating vascular disease. Stents are used toprovide support in a patient's hollow organs. To this end, stents of aconventional design have a filigree support structure composed ofmetallic struts; the support structure is initially present in acompressed form for insertion into the body, and is expanded at theapplication site. One of the main applications of stents of this type isto permanently or temporarily widen and hold open vasoconstrictions, inparticular constrictions (stenoses) of the coronary arteries. Inaddition, aneurysm stents are known, for example, which are used tosupport damaged vascular walls.

Many stents include a circumferential wall having a support force thatsuffices to hold the constricted vessel open to the desired extent; manystents also include a tubular base body through which blood continues toflow without restriction. The circumferential wall is typically formedby a latticed support structure that enables the stent to be inserted,in a compressed state having a small outer diameter, until it reachesthe constriction in the particular vessel to be treated, and to beexpanded there, for example using a balloon catheter, to the extent thatthe vessel finally has the desired, increased inner diameter.

The implant, in particular the stent, has a base body composed of animplant material. An implant material is a nonliving material that isused for a medical application and interacts with biological systems. Aprerequisite for the use of a material as an implant material that comesin contact with the body environment when used as intended is itsbiocompatibility. “Biocompatibility” refers to the capability of amaterial to evoke an appropriate tissue response in a specificapplication. This includes an adaptation of the chemical, physical,biological, and morphological surface properties of an implant to therecipient tissue, with the objective of achieving a clinically desiredinteraction. The biocompatibility of the implant material is furthermoredependent on the time sequence of the response of the biosystem in whichthe implant is placed. For example, irritations and inflammations, whichcan cause tissue changes, occur over the relative short term. Biologicalsystems therefore respond differently depending on the properties of theimplant material. Depending on the response of the biosystem, implantmaterials can be subdivided into bioactive, bioinert, anddegradable/resorbable materials.

Implant materials include polymers, metallic materials, and ceramicmaterials (as coating, for example). Biocompatible metals and metalalloys for permanent implants can contain, for example, stainless steels(e.g. 316L), cobalt-based alloys (e.g. CoCrMo casting alloys, CoCrMoforging alloys, CoCrWNi forging alloys, and CoCrNiMo forging alloys),pure titanium and titanium alloys (e.g. CP titanium, TiAl6V4 orTiAl6Nb7), and gold alloys. In the field of biocorrodible stents, theuse of magnesium or pure iron and biocorrodible base alloys of theelements magnesium, iron, zinc, molybdenum, and tungsten is proposed.

A biological response to polymeric, ceramic, or metallic implantmaterials depends on the concentration, duration of exposure, and typeof supply. The presence of an implant material often evokes inflammatoryresponses which can be triggered by mechanical irritations, chemicalsubstances, or metabolites. The inflammatory process is typicallyaccompanied by the immigration of neutrophil granulocytes and monocytesthrough the vascular walls, the immigration of lymphocyte effector cellswith the formation of specific antibodies to the inflammatory stimulus,activation of the complement system with the release of complementfactors which act as mediators, and, ultimately, activation of bloodcoagulation. An immunological response is usually closely associatedwith the inflammatory response and can lead to sensitization and thedevelopment of allergies. Known metallic allergens include nickel,chromium, and cobalt which are also used in many surgical implants asalloying constituents. A problem associated with the implantation of astent in a blood vessel is in-stent restenosis due to excessiveneointimal growth caused by a strong proliferation of arterial smoothmuscle cells and a chronic inflammatory response.

It is known that a greater level of biocompatibility can be achieved bycoating implant materials with particularly tissue-compatible materials.These materials are usually organic or synthetic-polymeric in nature andare partially of natural origin. Further strategies for preventingrestenosis focus on inhibiting proliferation using medication e.g.treatment using cytostatic agents. The active ingredients can beprovided e.g. on the implant surface in the form of a coating thatreleases an active ingredient.

It is furthermore known that the RGD triad (Arg-Gly-Asp) serves manyintegrins as a primary recognition site for proteins of theextracellular matrix. Peptides that contain this sequence can thereforemimic the ligands of these integrins and bind thereto.

Due to the fact that RGD peptides are selective antagonists forintegrins, their medical relevance—or the medical relevance ofpeptidomimetics derived therefrom—is the subject of research.

SUMMARY

The present invention provides an implant having a coating, wherein thecoating contains or is composed of a functionalized RGD peptidomimeticRGD-P1 having the formula (1):

and/ora functionalized RGD peptidomimetic RGD-P2 having the formula (2):

Q, T as well as U, V independently of one another stand for O or NH,whereby Q≠T and U≠v.A stand for O or (CH₂)₁₋₃ and X for CH₂, O, N or S, whereby if A standfor O, X must stand for CH₂.

Y stands for CH₂. O, N or S as well as Z and M independently of oneanother stand for O or S.

n or r stand for 1 till 6 and m stands for 2 till 8.

Furthermore P stands for a group selected of substituted orunsubstituted alkylen, preferably (CH₂)₁₋₆, or substituted orunsubstituted, aromatic or aliphatic N- or O-cycloalkylen.

R, independently of one another, stands for a polymeric group selectedfrom chitosan and polylysine, wherein polylysine comprises poly-D-lysineand poly-L-lysine; polylysine is preferably poly-L-lysine.

This aspect of the invention is based on the finding, for example, thatby using the coupling, according to the invention, to the polymerschitosan or polylysine, the functionalized RGD peptidomimetics that areobtained can be applied to an implant particularly advantageously as athin layer, while their integrin specificity and their integrin bindingcapabilities are substantially maintained. If an implant having anactive ingredient-releasing coating includes a layer containing orcomposed of the functionalized RGD peptidomimetic RGD-P1 according toformula (1) and/or RGD-P2 according to formula (2), e.g. in the form ofa cover layer, the elution characteristics of the underlying base forthe active ingredient is not changed or changed only to a minor extent.Some implant embodiments according to the invention is thereforecharacterized in that the adhesion of the implant is promoted by theintegrin binding of the functionalized RGD peptidomimetic, while noeffects—or only negligible effects—on the properties of underlyinglayers result.

DETAILED DESCRIPTION

Cyclic RGD peptides (cRGD) or RGD peptidomimetics can be used ascomponents of an implant coating. Loading polymer stents withintegrin-binding cRGD peptides can reduce neointimal hyperplasia byattracting endothelial progenitor cells.

In terms of developing implants coated with a coating that releasesactive ingredients, in particular stents coated with active ingredients(which are referred to as drug eluting stents or DES), the use of cRGDsor RGD peptidomimetics is a promising approach to improving thecompatibility of implants. The use of RGD peptidomimetics is desirableto improve the healing process. For longer-term developments, however,it can be desirable to release other active ingredients, e.g. rapamycin,from the coating. Adding RGD peptidomimetics subsequently to an existingcoating system therefore can create a problem related to ensuring thatthe RGD peptidomimetics are provided on the surface of the implant atthe earliest possible point in time, while ensuring that the elutioncharacteristic of an active ingredient contained in a base of thecoating underneath the surface does not change. Otherwise, the elutioncharacteristic of the active ingredient would have to be re-optimized,which is very difficult to do in isolated cases, and which makes itdifficult to vary the system.

With these considerations in mind, embodiments of the present inventionadvantageously design the provision of RGD peptidomimetics on thesurface of an implant such that the elution characteristic out the baselayer situated underneath do not change or change in only anunsubstantial manner, for an active ingredient embedded in an underlyingbase layer. Furthermore, in at least some invention embodiments therecognition sites of the RGD peptidomimetics are accessible immediatelyafter implantation, instead of their becoming exposed by the gradualdegradation of the coating matrix. These are only some of the benefitsand advantages achieved by embodiments of the invention.

An RGD peptidomimetic is understood to be a chemical compound thatbehaves substantially like the fundamental protein in terms of thebinding properties to selected integrins, but differs from thefundamental protein in terms of structure. Peptidomimetics typically donot have a continuous peptide backbone composed of peptide bonds and areusually composed non-exclusively of proteinogenic amino acids.

In the present case, the functionalized RGD peptidomimetic is RGD-P1having the formula (1) is a compound that has an integrin-binding domainthat preferably binds to a₅β₁ integrin. The integrin-binding domain ofRGD-P1 has inter alia the following structure:

The functionalized RGD peptidomimetic is RGD-P2 having the formula (2)is a compound that has an integrin-binding domain that preferably bindsto a_(v)β₃ integrin. The integrin-binding domain of RGD-P2 has interalia the following structure:

The integrin-binding domains of the RGD peptidomimetics RGD-P1 andRGD-P2 are each adjoined by an aminoalkanoic acid which is used as aspacer.

The RGD peptidomimetics RGD-P1 and RGD-P2 are functionalized on the freeamino group of the aminoalkanoic acid in that way, that it is covalentlybound via one of the free amino groups of the polymers to chitosan orpolylysine.

The functionalized RGD peptidomimetic that is obtained is therefore apolymer based on chitosan or polylysine, wherein a specific RGD-basedintegrin-binding domain is coupled to one, several, or all free aminogroups of chitosan or polylysine via coupling group isocyanate orthioisocyanate and an aminoalkanoic acid.

After implantation, to permit the most direct contact possible betweenthe integrin-binding domains of the RGD peptidomimetics of the implantto integrins of surrounding cells, the implant according to at leastsome invention embodiments has the coating containing the functionalizedRGD peptidomimetic RGD-P1 according to formula (1) and/or RGD-P2according to formula (2) as the cover layer. A “cover layer” isunderstood to mean a layer that separates the implant together with anyunderlying layer(s) from the surroundings, at least in sections, as theoutermost coating. According to a preferred embodiment, the cover layeroverlays on an exterior side the coating of the implant which has aninterior side that is adjacent to the base body of the implant.

Underneath the cover layer the implant can have a base layer that can becomposed of one or a plurality of different layers. The cover layercovers the base layer at least partially or entirely. The base layer caninclude a layer that releases an active ingredient.

According to the invention, a coating refers to the application, atleast in sections, of the components of the coating on the base body ofthe implant. Preferably, the coating covers the entire surface of thebase body of the implant. A layer (either the cover layer or a baselayer) thickness may be in the range of 1 nm to 100 μm, preferably 300nm to 15 μm, although other thicknesses including smaller than 1 nm andgreater than 100 μm may be used. The coating can be applied directly tothe surface of the implant. The processing can be performed usingstandard methods for the coating with examples including but not limitedto spraying, dipping, deposition, painting, and the like. The base layercan be composed of single-layered systems or multiple-layered systems(e.g. base coat layers, drug coat layers, or top coat layers). The baselayer can be applied directly to the base body of the implant, orfurther layers can be provided therebetween. Methods for coatingimplants, for creating the base layer, and for creating the cover layerare known to a person skilled in the art, and include but are notlimited to spraying, dipping, deposition, painting, and the like.

According to the invention, an active ingredient is a medicinal agenthaving a pharmaceutical effect, and which is used in the human body oranimal body to cure, alleviate, prevent, or detect illness. Activeingredients include paclitaxel, sirolimus, rapamycin, rapamycinderivatives, and others. Others include active ingredients that act viathe mTOR recognition site and RAS inhibitors, in particular those thatprevent RAS adhesion to the cell membrane.

The implant according to the invention may be a stent, particularlypreferably a biocorrodible stent. The stent can comprise a base bodythat contains or is composed of a biodegradable implant material. In thefield of biocorrodible stents, the use of magnesium or pure iron andbiocorrodible base alloys of the elements magnesium, iron, zinc,molybdenum, and tungsten may be made. In particular, the base body ofthe stent according to some invention embodiments can comprise or becomposed of a biocorrodible magnesium alloy.

In this context, an alloy is understood to be a metallic microstructurehaving magnesium, iron, zinc or tungsten as the main component. The maincomponent is the alloy component that comprises the largest weightcomponent of the alloy. A portion of the main component is preferablymore than 50% by weight, in particular more than 70% by weight.

The composition of the alloys of the elements magnesium, iron, zinc ortungsten can be selected such that they are biocorrodible. Within thescope of the invention, those alloys are referred to as beingbiocorrodible that degrade in a physiological environment, and thereforethe entire implant or the part of the implant composed of the materialloses its mechanical integrity. Artificial plasma is used as a testmedium to test the corrosion behavior of a potential alloy, theartificial plasma being specified according to EN ISO 10993-15:2000 forbiocorrosion tests (composition NaCl 6.8 g/l, CaCl₂ 0.2 g/l, KCl 0.4g/l, MgSO₄ 0.1 g/l, NaHCO₃ 2.2 g/l, Na₂HPO₄ 0.126 g/l, NaH₂PO₄ 0.026g/l). To perform the test, a sample of the alloy to be investigated isstored in a closed sample container with a defined quantity of the testmedium at 37° C. Samples are taken at certain time intervals, which arebased on the anticipated corrosion behavior, of a few hours to severalmonths, and they are examined in a known manner for traces of corrosion.The artificial plasma according to EN ISO 10993-15:2000 corresponds to ablood-like medium and therefore provides a way to reproducibly adjust aphysiological environment within the scope of the invention.

In many suitable biocorrodible metallic implant materials, the maincomponent is an element of the group alkaline metals, alkaline-earthmetals, iron, zinc, and aluminium. Alloys based on magnesium, iron andzinc are described as being particularly suitable. Minor constituents ofthe alloys can be manganese, cobalt, nickel, chromium, copper, cadmium,lead, tin, thorium, zirconium, silver, gold, palladium, platinum,silicon, calcium, lithium, aluminium, zinc and iron. Furthermore, onesuitable biocorrodible magnesium alloy has a portion of magnesium >90%,yttrium 3.7-5.5%, rare-earth metals 1.5-4.4% and the rest <1%, which issuitable in particular for manufacturing a stent, with one example stentin the form of a self-expanding or balloon-expandable stent.

A further aspect of the invention is the provision of a method formanufacturing an aforementioned implant. The method comprises the steps:

-   -   Coat a stent with a layer that contains or is composed of        chitosan and/or polylysin;    -   Covalent coupling of a primary amine contained in chitosan        and/or polylysine with the precursor of the RGD peptidomimetic        RGD-P1 having the formula (3):

and/ora precursor of the RGD peptidomimetic RGD-P2 having the formula (4):

Q, T as well as U, V independently of one another stand for O or NH,whereby Q≠T and U≠V.A stand for O or (CH₂)₁₋₃ and X for CH₂, O, N or S, whereby if A standfor O, X must stand for CH₂.Y stands for CH₂. O, N or S as well as Z and M independently of oneanother stand for O or S.n or r stand for 1 till 6 and m stands for 2 till 8.

Furthermore P stands for a group selected of substituted orunsubstituted alkylen, preferably (CH₂)₁₋₆, or substituted orunsubstituted, aromatic or aliphatic N- or O-cycloalkylen.

Using the proposed method, it is possible e.g. to also coat existingactive ingredientreleasing implants, stents, or systems with a coverlayer containing the functionalized RGD peptidomimetics RGD-P1 and/orRGD-P2 after they are coated with the active ingredient itself.

A particular advantage is that the active ingredients that have alreadybeen coated onto the surface of the active ingredient-releasing implantcan be provided with the coating containing the compounds according tothe invention while maintaining their biological activity. Prior methodsknown to a person skilled in the art for coating a stent that has beenpreviously coated (with sirolimus, by way of example) result in apartial destruction of the active ingredient, thereby reducing thebiological activity of the implant.

The coating of the implant according to embodiments of the inventionwith a functionalized RGD peptidomimetic can be performed as follows.Chitosan or polylysine can be applied in a diluted aqueous solution, forexample to a stent that may have also been precoated with a basematerial containing an active ingredient, using a spraying process or animmersion process. Subsequent coupling with precursors of the RGDpeptidomimetic RGD-P1 and/or RGD-P2 takes place in the aqueous medium;when lipophilic active ingredients are used, there is no risk that theactive ingredients bound in the base will elute in this medium andthereby lower the load of active ingredient.

In a further aspect, embodiments of the present invention providecompounds having:

in whichQ, T as well as U, V independently of one another stand for O or NH,whereby Q≠T and U≠V.

A stand for O or (CH₂)₁₋₃ and X for CH₂, O, N or S, whereby if A standfor O, X must stand for CH₂;

Y stands for CH₂. O, N or S as well as Z and M independently of oneanother stand for O or S;n or r stand for 1 till 6 and m stands for 2 till 8;P stands for a group selected of substituted or unsubstituted alkylen,preferably (CH₂)₁₋₆, or substituted or unsubstituted, aromatic oraliphatic N- or O-cycloalkylen; andR, independently of one another, stands for a polymeric group selectedfrom chitosan and polylysine, wherein polylysine comprises poly-D-lysineand poly-L-lysine; polylysine is preferably poly-L-lysine.

The aforementioned compounds according to embodiments of the inventioncan be used, for example, in a coating of an implant, in particular inthe cover layer of an active ingredient-releasing stent, wherein thestent comprises a resorbable or permanent base body composed of ametallic or polymeric material with one or more layers under the coverlayer.

Some aspects of the invention is explained below in greater detail belowwith reference to embodiments.

Reaction of the Compound Having the Formula (3) and (4) on a ChitosanSurface

Spray a stent comprising a coating of polylactide (PLLA) and sirolimuswith a solution of chitosan in diluted acetic acid (0.3%). After drying,immerse the stent in a buffered (phosphate buffer 50 mM) aqueoussolution of a precursor of the functionalized RGD peptidomimetic RGD-P1having the formula (3) or a precursor of the functionalized RGDpeptidomimetic RGD-P2 having the formula (4) (concentration: 5 mg/ml; pH7; room temperature). After 1 hour, all amine groups of the chitosanhave reacted with the reactive groups of the coupling groups.

The stent does not exhibit deviating elution kinetics for sirolimus. Incontrast to stents that have been coated solely with PLLA and sirolimus,a first endothelial covering is visible just three days afterimplantation in the animal.

It will be apparent to those skilled in the art that numerousmodifications and variations of the described examples and embodimentsare possible in light of the above teaching. The disclosed examples andembodiments are presented for purposes of illustration only. Therefore,it is the intent to cover all such modifications and alternateembodiments as may come within the true scope of this invention.

1. An implant having a coating, wherein the coating comprises afunctionalized RGD peptidomimetic RGD-P1 having the formula (1):

and/or a functionalized RGD peptidomimetic RGD-P2 having the formula(2):

wherein Q, T as well as U, V independently of one another stand for O orNH, whereby Q≠T and U≠V; A stand for O or (CH₂)₁₋₃ and X for CH₂, O, Nor S, whereby if A stand for O, X must stand for CH₂; Y stands for CH₂,O, N or S as well as Z and M independently stand for O or S; n or rstand for 1 till 6 and m stands for 2 till 8; P stands for a groupselected of substituted or unsubstituted alkylen, preferably (CH₂)₁₋₆,or substituted or unsubstituted, aromatic or aliphatic N- orO-cycloalkylen; and R, independently of one another, stands for apolymeric group selected from chitosan and polylysine, whereinpolylysine comprises poly-D-lysine and poly-L-lysine; polylysine ispreferably poly-L-lysine.
 2. The implant according to claim 1, whereinthe coating is a cover layer.
 3. The implant according to claim 2, andfurther comprising a base layer under the cover layer.
 4. The implantaccording to claim 3, wherein the base layer includes a layer thatreleases an active ingredient.
 5. The implant according to claim 4, inwhich the active ingredient is one or more of paclitaxel, sirolimus, andrapamycin.
 6. The implant according to claim 1, wherein the implant isone of a stent, a sensor, and an electrode.
 7. The implant according toclaim 6, wherein the stent comprises a resorbable base body composed ofa polymeric material.
 8. A method for manufacturing an implant accordingto claim 1, wherein the method comprises the steps: Coat a stent with alayer that comprises one or more of chitosan and polylysin; Covalentcoupling a primary amine contained in the one or more of chitosan andpolylysine with the coupling group of one or more of a precursor of aRGD peptidomimetic RGD-P1 having the formula (3):

and a precursor of the functionalized RGD peptidomimetic RGD-P2 havingthe formula (4):

wherein Q, T as well as U, V independently of one another stand for O orNH, whereby Q≠T and U≠V; A stand for O or (CH₂)₁₋₃ and X for CH₂, O, Nor S, whereby if A stand for O, X must stand for CH₂; Y stands for CH₂.O, N or S as well as Z and M independently of one another stand for O orS; n or r stand for 1 till 6 and m stands for 2 till 8; and P stands fora group selected of substituted or unsubstituted alkylen, preferably(CH₂)₁₋₆, or substituted or unsubstituted, aromatic or aliphatic N- orO-cycloalkylen.
 9. A compound according to:

in which Q, T as well as U, V independently of one another stand for Oor NH, whereby Q≠T and U≠V; A stand for O or (CH₂)₁₋₃ and X for CH₂, O,N or S, whereby if A stand for O, X must stand for CH₂; Y stands forCH₂. O, N or S as well as Z and M independently of one another stand forO or S; n or r stand for 1 till 6 and m stands for 2 till 8; P standsfor a group selected of substituted or unsubstituted alkylen, preferably(CH₂)₁₋₆, or substituted or unsubstituted, aromatic or aliphatic N- orO-cycloalkylen; and R, independently of one another, stands for apolymeric group selected from chitosan and polylysine, whereinpolylysine comprises poly-D-lysine and poly-L-lysine; polylysine ispreferably poly-L-lysine.
 10. The use of a compound according to claim 9in a coating of an implant, in particular in the cover layer of a stentthat releases an active ingredient.
 11. The implant according to claim6, wherein the stent comprises a permanent base body composed of ametallic material.
 12. An implant according to claim 1, wherein thelayer is a cover layer, and further comprising a base layer underlyingthe cover layer, an active ingredient embedded in the base layer; and,wherein the elution characteristic of the active ingredient in the baselayer underneath the cover layer do not change with the presence of thecover layer as compared to having no cover layer present.
 13. An implantas defined by claim 12 wherein the active ingredient is a rapamycinderivative.
 14. An implant as defined by claim 1 wherein the cover layerthickness is between about 300 nm to 15 μm.
 15. An implant as defined byclaim 1 wherein the recognition sites of the RGD peptidomimetics areaccessible immediately after implantation.